Our previous findings have shown that ethinyl estradiol (EE) is a strong promoter of hepato-carcinogenesis. Our more recent mechanistic studies have revealed that EE causes an increase in a serum hepatocyte growth factor, possibly in the EGF family, and that EE pretreatment dramatically potentiates the responsiveness of cultured hepatocytes to subsequent treatment with EGF. In this application we propose to test the hypothesis that EE acts as a co-mitogen on hepatocytes by enhancing their responsiveness to a growth factor which works through the EGF receptor pathway. We will focus on the events which occur after the addition of EGF to EE pretreated cells which may contribute to the potentiation of EGF responsiveness. The specific aims are: 1) to determine the mechanism of potentiation of EGF responsiveness by EE in primary cultures of rat hepatocytes. The 2- fold increase in hepatocyte EGF receptor levels induced by EE may not be sufficient to account for the dramatic potentiation of EGF-induced DNA synthesis. We plan to determine whether one or more of the components of the EGF signal transduction pathway is affected by EE pretreatment; 2) to develop a continuous liver cell culture model which exhibits an EE-induced potentiation of EGF responsiveness in a manner similar to that observed in primary hepatocytes. The development of this model is desirable for studies on the effects of EE on EGF responsiveness in initiated cells (see below). We will initially use the well characterized WB liver cell line and determine whether these cells are positive for estrogen receptors (estr). If not, we will introduce the estr gene in an expression vector by transfection or by electroporation. We will then characterize the response of these cells to EGF following EE treatment; 3) to determine the effect of EE on EGF responsiveness in initiated cells. We have shown in vivo that liver DNA synthesis in response to EE is transient and returns to baseline levels even with continuous EE treatment. However, in DEN treated rats, initiated hepatocytes must continue to grow in order to give rise to preneoplastic foci. Here we will focus on determining the mechanism(s) by which initiated cells are altered in their growth response to the continuous presence of EE. We will initiate EE/EGF responsive liver cell lines (see above) by transfection with activated oncogenes under an inducible promoter (e.g. MT-ras, MT-myc and MT-raf) and determining the effect of the expression of these genes on EE/EGF responsiveness. We will also determine whether hepatocytes isolated from GGT foci are altered in their responsiveness to EE/EGF compared to normal hepatocytes; and 4) to characterize further the serum factor(s) enhanced by E treatment in vivo initially by determining whether this factor is novel or the same as HPTA, recently purified and characterized by Michalopoulos and co-workers. These studies should provide new information on the mechanisms of both liver growth stimulation and tumor promotion by EE.